Harmful algal blooms (HABs) can have severe ecological, economical, and human health impacts. At least 32 states have reported human or mammal exposures to HAB toxins. Furthermore, studies suggest that the frequency and intensity of HAB events have increased over the past few decades and will continue to increase this century. Knowledge gaps remain regarding the ecological drivers of these events which have hindered the development of successful mitigation strategies. Most HABs are comprised of several genera with each species containing toxic and non-toxic strains. This has made understanding the ecology of these events using traditional methods extremely difficult. Advances in molecular techniques (e.g. quantitative PCR) have facilitated the first steps towards a deeper understanding of these complex events although previous studies have generally focused on a single organism. The investigators' goal is to develop and employ a flexible real-time multiplex quantitative polymerase chain reaction (MqPCR) assay for the simultaneous quantification of co-occurring toxic HAB genera. This will facilitate the next important step in furthering our understanding of these complex events and will significantly improve the integration of HAB activities into existing state and federal monitoring and observational programs. The investigators have four primary objectives: 1) Develop MqPCR specific TaqMan primers and probes for the detection of anatoxin-a producing cyanobacteria as well as toxic eugleniods to incorporate into the existing MqPCR method. 2) Design and field validate a flexible MqPCR that can be tailored to specific HAB events. By designing both TaqMan primers and probes to be replicated in the same thermal cycling program, they will be able to select specific primer sets to match different bloom situations. 3) The investigators will use the MqPCR to investigate the response of microcystin/nodularin-, cylindrospermopsin-, saxitoxin-, and anatoxin-a-producing cyanobacteria as well as potentially toxic euglenoids to environmental variables individually, as well as in relation to each other, over the course of a bloom. They will sample from four sites across the nation each year that routinely develop dense blooms of mixed genera; western Lake Erie, Grand Lake St. Marys, OH, St. Johns River, FL, and Nueces River, TX. Samples will be collected weekly before, during and after bloom events (150 field samples minimum). The investigators will also collect nutrient data, physiochemical parameters and algal toxin data. 4) Identify the role of poorly understood toxin-producing algae with U.S. freshwaters. The three investigators (2 faculty members, one post-doctoral researcher) and a co-investigator's vast expertise in a number of disciplines (microbiology, genetics, and chemistry) will allow for a multi-disciplinary approach toward this research. The MqPCR technique developed will be a valuable tool for the rapid monitoring of HAB genera that can be applied across the marine- freshwater continuum. This study will provide important data for the modeling and prediction of future HAB events as well as the development of early warning networks to protect humans from exposure to HAB toxins. Public Health Relevance: Algal blooms cause serious water quality and human health issues; however, their ecology remains poorly understood. Using a cutting-edge genetic method the investigators will further their understanding of the environmental factors promoting these toxic algae. This will allow for the further development of successful predictive models and algal remediation strategies that will help protect humans from the negative human health effects of algal toxin exposure.